In known processes of interaction analysis, biomolecular interaction is studied with receptor-ligand systems wherein the receptor is usually a biomacromolecule (e.g. a protein or single-stranded DNA) and the ligand is a “probe”, a molecule usually of low molecular weight and of biological or synthetic origin (peptides, oligonucleotides or what are referred to as small organic molecules). Such ligands exhibit highly specific structural properties which can interact with a receptor provided it exhibits corresponding structures. The binding to the receptor can take place via one or several ligands. Furthermore, systems are studied wherein both interaction partners are macromolecules (e.g. protein-protein interaction).
Interaction analysis is used in the pharmaceutical and agrochemical industries for researching active substances. The goal is to analyze as large a number of different samples as possible in as short a time period as possible. There is a growing interest to develop testing systems that are able to simultaneously subject a large number of both identical and different molecules to a certain test method for detecting their biospecific binding behavior in order to identify molecules with a specific effect (High-Throughput Screening, HTS). Such a parallelization is closely linked to a simultaneous miniaturization of the testing arrangement and automation of the testing procedure. Furthermore, interaction analysis is used in the research of the genome (polymorphisms (SNP) analysis or expression pattern analysis) and also in food analysis.
It constitutes a practical advantage to bind one of the interaction or binding partners, i.e. the ligand or the receptor, to an organic or inorganic surface, thereby imparting bioactivity to that surface due to the generation of a specific boundary layer. The bond is preferably a covalent bond or a bond formed by adsorption. The immobilization of an interaction partner facilitates the process, such as for example carrying out washing steps, and in combination with a suitable, most of the time optical, detection process (e.g. fluorescence measurement) it may provide information on the presence and degree of interaction between the interaction partners on a molecular level. Comparability and reproducibility of the results are important prerequisites for the interpretation of such data stemming from the interaction between receptor and ligand.
Bioactive surfaces can be generated on different organic and inorganic carriers, such as for example cellulose, silicates or noble metal surfaces. In addition, these carriers can be components of a sensor system. As regards the bioactive boundary layer, films consisting of organic monolayers (Bain and Whitesides, Angew. Chem. 101 [Applied chemistry 101] (1989) 522-8; Zhong and Porter, Anal. Chem. [Analytical chemistry] (1995) 709A-715A) are especially advantageous (physicochemical stability, structural uniformity). Processes are known for preparing such films, wherein for example in a first step alkyl thiols are chemisorbed onto gold. The long-chain molecules assemble on the solid phase as a highly ordered monolayer (self-assembled monolayer, SAM), whereby the gold atoms are complexed by the sulfur functions. Short-chain alkyl thiols however, such as butane thiol, do not form such highly ordered monolayers (R. Berger, et al., Science 27 (1997), 2021-2024). Such SAMs, which in the above-mentioned case do not yet exhibit any specific bioactive structures, are known in the prior art and have been appropriately characterized by means of many physical methods. In Poirier and Pylant, Science, 272 (1996) 1145-8, scanning tunneling microscopic photographs of such monolayers on gold can be found. Apart from gold, other metals as well can serve as the solid phase for such monolayers.
The immobilization of an interaction partner imparting bioactivity to the surface, in particular to a sensor surface, can be effected by so-called anchors. A suitable anchor molecule comprises at least two functional units present at opposite ends of the anchor and allowing binding to the solid phase surface on the one hand and binding to the interaction partner to be immobilized on the other hand. The latter will be referred to in the following as “head group”. The nature of the structural element connecting these functional groups should preferably be such that it allows at least the formation of a highly ordered monolayer on the basis of the anchor molecules.
The generation of a bioactive surface using such anchors can be carried out in one or several steps. In the single-step process, the interaction partner is bonded to the head group of the anchor prior to its immobilization. Suitable anchors for the single-step process are described in DE 199 24 606.8. For providing a measuring surface, a complete ligand-anchor conjugate (LAC) is applied onto the surface. This is a molecule connecting the interaction partner to be immobilized and the functional group necessary for binding to the surface by means of a structure capable of forming an SAM.
In multi-step processes, first a binding surface in the form of an organic boundary layer is generated which does not yet exhibit the desired specific structural features, however, which is suitable for binding or can be activated to bind the interaction partners to be immobilized, e.g. ligands. Thus, immobilization of an interaction partner takes place after the generation of a first boundary layer that is not bond-specific by means of a covalent, ionic or complex bond via the head group of the binding component. For this purpose, the head group can be used directly or after it has been activated. Examples of such head groups are described in EP 485 874 A2.
An important advantage of the multi-step process is the fact that the same surface can be used for every interaction partner to be immobilized. Therefore, the resulting concentration of the interaction partners to be immobilized is the same on the surface as well, which is not or not always a given in the single-step process. This is particularly important for the comparability and reproducibility of the different measuring results. However, it also has to be kept in mind that the chemical homogeneity of the non-biospecific binding surface decreases again as the number of potentially necessary activation steps increases.
A step-by-step formation of a diluted binding layer is described in WO 98/40739 A1, wherein in a first step cystamine is applied onto a gold surface and the provision of maleimide as head group is realized in a second chemical reaction. The disadvantage of this process is that another activation step is required for obtaining a binding surface.
Furthermore, reactive amino functions remain that can be responsible for an unspecific binding of macromolecules such as proteins.
It has to be made sure in all multi-step processes that the reaction binding the interaction partner to be immobilized to the anchor via the head group proceeds selectively and in a controlled manner. In this connection, covalent binding of the interaction partner to be immobilized is advantageous since the bioactive surface generated in this manner is chemically more stable, for example with respect to regeneration or storage conditions that can be applied. These advantages particular apply in comparison with the non-covalent binding system streptavidin/biotin which is used frequently.
In particular in the case of immobilization of a small ligand, for instance a so-called “small molecule”, the selectivity and the quantitative course of the reaction is of utmost importance for the quantitative evaluation of binding studies or interaction analyses. Here, in contrast to e.g. macromolecular recognition structures, slight differences in the binding strength of the ligand to the receptor often have to be resolved. A ligand of higher affinity offered at a lower density (concentration) than another ligand with a lower binding affinity can lead to an artificially reduced signal, while the other way round a weaker ligand offered at a higher density (concentration) can lead to an artificially increased signal compared to another ligand.
In order to ensure a high degree of selectivity and the possibility of quantitative reactions, reactions with molecules carrying a thiol group or modified by means of a thiol group are employed in biochemistry. The reaction partner is often a macromolecule carrying a maleimidyl group. The product of the reaction of the thiol and the maleimidyl group is a thio succinimide (Michael addition) (G. T. Hermanson, Bioconjugate Techniques, Academic Press 1996, 229-252; Suda et al. Biochem. Biophys. Res. Comm. (1999), 261, 276-282).
A number of reaction partners for the thiol group are known from the prior art (referred to in the following as mercaptophiles), which also allow its selective and quantitative reaction. In addition to maleimides, they include the following compounds:
Iodo- and bromoacetamides, pyridyldithio compounds (G. T. Hermanson, Bioconjugate Techniques, Academic Press 1996, 229-252; C. Boeckler et al., J. Immun. Meth. (1996) 191, 1-10), Michael acceptors in general, acrylic acid derivatives such as the esters, amides, lactones or lactames thereof (K. Matsuura et al. J. Mass Spectrom. (1998) 33, 1199-1208; W. Adam et al. J. Org. Chem. (1995) 60, 578-584), methylene-gem-difluorocyclopropanes (T. Taguchi et al., Tetrahedron (1997). 53, 9497-9508), .alpha.,.beta.-unsaturated aldehydes and ketones (Chen et al., J. Am. Chem. Soc. (1994) 116, 2661-2662) and .alpha.,.beta.-unsaturated sulfones and sulfonamides.
The thiol/mercaptophile system offers the essential advantage that the binding of the interaction partner to be immobilized can be carried out under gentle reaction conditions (ambient temperature, neutral pH, buffer solutions can be used). This is of particular importance when unstable compounds or proteins that denature easily are used. Another advantage is that compared to carboxylic acid, amine or amide groups, a thiol functionality hardly ever occurs in active substances. Thus, an undesired reaction of mercaptophiles that may remain after the immobilization of one interaction partner and the target can largely be avoided.
The thiol/maleimide system is furthermore particularly known for its high degree of selectivity compared to other functionalities such as hydroxyl, amine, carboxyl or hydroxylamine groups. In addition, the formation of the covalent bond is characterized by a high reaction rate (cf. Schelt et al., Bioconj. Chem. (2000), 11, 118-123).
Although such reactions have proven successful in bioconjugate chemistry, anchors comprising both a thiol group for binding to the solid phase surface and a mercaptophilic head group for immobilizing an interaction partner are not disclosed in the prior art since both during synthesis and during the generation of the binding surface both intra- and intermolecular side reactions, and even polymerization, may occur (with respect to the purposeful polymerization of oligothiols with bis-maleimides, cf. L. R. Dix et al., Eur. Polym. J. 31 (1995), 653-658).
EP 485 874 A2 refers to this problem, which is avoided by the exclusive use of disulfide and sulfide groups in the anchor, in order to use for example maleimide as head group for the immobilization of proteins (reaction with —SH group of a Fab′ fragment). EP 698 787 A1 as well uses short-chain, maleimide-carrying disulfide anchors for the immobilization of proteins. However, anchors based on disulfide and sulfide groups show the disadvantage that an undesired spatial proximity of the head groups is generated. In this case, the head groups can interfere with each other in the immobilization reaction.
In order to be able to control the spatial proximity of adjacent ligands, EP 485 874 A2 and EP 698 787 A1 do not only apply anchor molecules onto the solid phase surface. Rather, the anchor molecules are “diluted”. This is due to the fact that if one partner, for example a ligand is bound to a solid phase carrier for interaction analysis, adjacent ligands on the surface can interfere with each other or with the interaction between the adjacent ligand and the interaction partner to be detected. Whitesides and his colleagues were able to verify this effect in the biospecific adsorption of carbonic anhydrase on benzosulfonamide groups, which were immobilized by means of alkane thiolates on gold, by demonstrating that the degree of undesired irreversible binding of carbonic anhydrase to a mixture of ligand-carrying anchor (immobilized sulfonamide) and diluting component (12,15,18-trioxa-20-hydroxyicosan-1--thiol) decreases as the amount of diluting component in the mixture increases (J. Am. Chem. Soc. 1995, 117, 12009-10).
In order to avoid this disadvantage, mixed surfaces—as shown in FIG. 1—can be applied, which are composed of ligand-carrying anchor molecules and so-called diluting molecules that do not carry any ligands and thus dilute the measuring surface.
The structural nature of the diluting component has to meet the prerequisite that it will not influence the interaction of the immobilized interaction partner and the free interaction partner. In particular, if possible, no specific or unspecific binding between the free interaction partner and the diluting component should occur (e.g. diluent with as high a resistance to protein adsorption as possible). Furthermore, the anchor molecule and the diluting component should be as structurally similar as possible to ensure that their mixing behavior on the solid phase surface is as homogeneous as possible.
On principle, prior art anchors that are based on disulfides and sulfides and thus present two head groups cannot guarantee a stochastic distribution of head-group-carrying molecules and diluting molecules. However, this is necessary in order to ensure a purposeful interaction of the bioactive surface with the free interaction partner later on.